Immune suppression in host animals is an essential precondition for xenograft success because the host immune system has a tendency to reject implanted cancer cells. We developed a novel zebrafish model based on two-round cancer cell xenotransplantation that achieved cancer-specific immunological tolerance without immunosuppression. We irradiated human cancer cells to abolish their proliferative abilities and implanted them into young zebrafish. These cells lasted for 2 weeks. Three months after the first xenotransplantation, the same zebrafish were implanted with nonirradiated cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. We conducted dual RNA-seq of the tumour with its surrounding tissues and identified several regulatory genes in zebrafish that are involved in immunity were altered with immunotolerance. In conclusion, this xenograft method has potential as a platform for anti-cancer drug discovery.